Subtilisins modified at position 225 resulting in a shift in catalytic activity

ABSTRACT

There are described certain subtilisins wherein the amino acid sequence of such subtilisins has been modified at a position equivalent to +225 in Bacillus amyloliquefaciens, such that an amino acid selected from the group consisting of alanine, leucine, methionine, glutamine, valine, and serine, has been substituted for the amino acid residue naturally occurring at such position.

Cross-reference is made to U.S. patent application Ser. No. 084,589filed Aug. 12, 1987 (pending), which is a continuation-in-part of U.S.patent application Ser. No. 035,652 filed Apr. 6, 1987 (abandoned),which is a continuation-in-part of U.S. patent application Ser. No.858,594 filed Apr. 30, 1986 (abandoned), which is a continuation-in-partof U.S. patent applications Ser. Nos. 614,612 (issued), 614,615(abandoned), 614,617 (abandoned) and 614,491 (abandoned), all filed May29, 1984, each of which are incorporated herein by reference.Application Ser. No. 614,612 issued as U.S. Pat. No. 4,760,025 on Jul.26, 1988.

FIELD OF THE INVENTION

The invention relates to novel carbonyl hydrolase mutants derived fromthe amino acid sequence of naturally-occurring or recombinant non-humancarbonyl hydrolases and to DNA sequences encoding the same. Such mutantcarbonyl hydrolases, in general, are obtained by in vitro modificationof a precursor DNA sequence encoding the naturally-occurring orrecombinant carbonyl hydrolase to encode the substitution, insertion ordeletion of one or more amino acids in a precursor amino acid sequence.

BACKGROUND OF THE INVENTION

Serine proteases are a subgroup of carbonyl hydrolase. They comprise adiverse class of enzymes having a wide range of specificities andbiological functions. Stroud, R. M. (1974) Sci Amer. 131, 74-88. Despitetheir functional diversity, the catalytic machinery of serine proteaseshas been approached by at least two genetically distinct families ofenzymes: the Bacillus subtilisin-type serine proteases and the mammalianand homologous bacterial trypsin-type serine proteases (e.g., trypsinand S. gresius trypsin). These two families of serine proteases showremarkably similar mechanisms of catalysis. Kraut, J. (1977) Ann. Rev.Biochem. 46, 331-358. Furthermore, although the primary structure isunrelated, the tertiary structure of these two enzyme families bringtogether a conserved catalytic triad of amino acids consisting ofserine, histidine and aspartate.

In subtilisin-type serine proteases, the OG group of the catalytic sidechain of serine (serine-221 in subtilisin) is located near aminoterminus of a long central α-helix which extends through the molecule.Bott, et al. (1988) J. Biol. Chem. 263, 7895-7906. In Bacillusamyloliquifaciens subtilisin this α-helix comprises alanine 223 throughlysine 237. This helix is conserved in evolutionarily relatedsubtilisin-type serine proteases but is not found in the catalytic sitesof trypsin-type serine proteases.

McPhelan, et al. (1988) Biochemistry 27, 6582-6598. The α-helixassociated with the active site of subtilisin-type serine proteases hasled to the suggestion that the dipole of this helix may have afunctional role in catalysis. Hol, W. G. J. (1985) Prog. Boiphys. Molec.Biol. 45, 149-195. The lack of α-helix at the active site of thetrypsin-type serine proteases, however, has raised the unresolvedquestion of whether the active site helix of subtilisin-type serineproteases is of any significance in catalysis. Hol (1985) supra.

Subtilisin is a serine endoprotease (MW27,500) which is secreted inlarge amounts from a wide variety of Bacillus species. The proteinsequence of subtilisin has been determined from at least four differentspecies of Bacillus. Markland, F. S., et al. (1971) in The Enzymes, ed.Boyer, P. D., Acad Press, New York, Vol. III, pp. 561-608; Nedkov, P. etal. (1983) Hoppe-Seyler's Z. Physiol. Chem. 364, 1537-1540. Thethree-dimensional crystallographic structure of subtilisin BPN' (from B.amyloliquefaciens) to 2.5 Å resolution has also been reported. Bott(1988) supra: McPhelan (1988) supra: Wright, C. S., et al. (1969) Nature221, 235-242; Drenth, J. et al. (1972) Eur. J. Biochem. 26, 177-181.These studies indicate that although subtilisin is genetically unrelatedto the mammalian serine proteases, it has a similar active sitestructure. The x-ray crystal structures of subtilisin containingcovalently bound peptide inhibitors (Robertus, J. D., et al. (1972)Biochemistry 2439-2449), product complexes (Robertus, J. D., et al.(1972) Biochemistry 11, 4293-4303), and transition state analogs(Matthews, D. A., et al (1975) J. Biol. Chem. 250, 7120-7126; Poulos, T.L., et al. (1976) J. Biol. Chem. 251, 1097-1103), which have beenreported have also provided information regarding the active site andputative substrate binding cleft of subtilisin. In addition, a largenumber of kinetic and chemical modification studies have been reportedfor subtilisin (Philipp, M., et al. (1983) Mol. Cell. Biochem. 51, 5-32;Svendsen, I. B. (1976) Carlsberg Res. Comm. 41, 237-291; Markland, F. S.Id.). In one report the side chain of methione at residue 222 ofsubtilisin was converted by hydrogen peroxide to methionine-sulfoxide(Stauffer, D. C., et al. (1965) J. Biol. Chem. 244, 5333-5338). Inanother, subtilisin was chemically modified to thiosubtilisin (Polgar,L. et al (1981) Biochem. Biophys. Acta. 667, 351-354). Based on theanalysis of peptide fragments, the authors suggest that the chemicalmodification of subtilisin to thiosubtilisin caused the modification ofserine at position 221 to cysteine.

Substrate specificity is a ubiquitous feature of biologicalmacromolecule that is determined by chemical forces including hydrogenbonding, electrostatic, hydrophobic and stearic interactions. Jencks, W.P., in Catalysis in Chemistry and Enzymology (McGraw-Hill, 1969) pp.321-436; Fersht, A., in Enzyme Structure and Mechanism (Freeman, SanFrancisco, 1977) pp. 226-287. Substrate specificity studies of enzymes,however, have been limited to the traditional means of probing therelative importance of these binding forces. Although substrate analogscan be synthesized chemically, the production of modified enzyme analogshas been limited to chemically modified enzyme derivatives (Kaiser, E.T., et al. (1985) Ann. Rev. Biochem. 54, 565-595 and naturally occurringor induced mutants (Kraut, J. (1977) Ann. Rev. Biochem. 46, 331-358;Paterson, A. et al. (1979) J. Gen. Micro. 114, 65-85; Uehara, H. et al.(1979) J. Bacteriology 139, 583-590; Kerjan, P. et al. (1979) Eur. J.Biochem. 98, 353-362).

The recent development of various in vitro techniques to manipulate theDNA sequences encoding naturally-occurring polypeptides as well asrecent developments in the chemical synthesis of relatively shortsequences of single and double stranded DNA has resulted in thespeculation that such techniques can be used to modify enzymes toimprove some functional property in a predictable way. Ulmer, K. M.(1983) Science 219, 666-671. The only working example disclosed therein,however, is the substitution of a single amino acid within the activesite of tyrosyl-tRNA synthetase (Cys35Ser) which lead to a reduction inenzymatic activity. See Winter, G., et al. (1982) Nature 299, 756-758;and Wilkinson, A. J., et al. (1983) Biochemistry 22, 3581-3586 (Cys35Glymutation also resulted in decreased activity).

When the same t-RNA synthetase was modified by substituting a differentamino acid residue within the active site with two different aminoacids, one of the mutants (Thr51Ala) reportedly demonstrated a predictedmoderate increase in kcat/Km whereas a second mutant (Thr51Pro)demonstrated a massive increase in kcat/Km which could not be explainedwith certainty. Wilkinson, A. H., et al. (1984) Nature 307, 187-188.

Another reported example of a single substitution of an amino acidresidue is the substitution of cysteine for isoleucine at the thirdresidue of T4 lysozyme. Perry, L. J., et al. (1984) Science 226,555-557. The resultant mutant lysozyme was mildly oxidized to form adisulfide bond between the new cysteine residue at position 3 and thenative cysteine at position 97. This cross-linked mutant was initiallydescribed by the author as being enzymatically identical to, but morethermally stable than, the wild type enzyme. However, in a "Note Addedin Proof", the authors indicated that the enhanced stability observedwas probably due to a chemical modification of cysteine at residue 54since the mutant lysozyme with a free thiol at Cys54 has a thermalstability identical to the wild type lysozyme.

Similarly, a modified dehydrofolate reductase from E. coli has beenreported to be modified by similar methods to introduce a cysteine whichcould be crosslinked with a naturally-occurring cysteine in thereductase. Villafranca, D. E., et al. (1983) Science 222, 782-788. Theauthors indicates that this mutant is fully reactive in the reducedstate but has significantly diminished activity in the oxidized state.In addition, two other substitutions of specific amino acid residues arereported which resulted in mutants which had diminished or no activity.

As set forth below, several laboratories have also reported the use ofsite directed mutagenesis to produce the mutation of more than one aminoacid residue within a polypeptide.

The amino-terminal region of the signal peptide of the prolipoprotein ofthe E. coli outer membrane was stated to be altered by the substitutionor deletion of residues 2 and 3 to produce a charge change in thatregion of the polypeptide. Inoyye, S., et al. (1982) Proc. Nat. Acad.Sci. USA 79, 3438-3441. The same laboratory also reported thesubstitution and deletion of amino acid residues 9 and 14 to determinethe effects of such substitution on the hydrophobic region of the samesignal sequence. Inouye, S., et al. (1984) J. Biol. Chem. 259,3729-3733. In the case of mutants at residues 2 and 3 the authors statethat the results obtained were consistent with the proposed loop modelfor explaining the functions of the signal sequence. However, asreported the mutations at residues 9 and 14 produced results indicatingthat the signal peptide has unexpended flexibility in terms of therelationship between its primary structure and function in proteinsecretion.

Double mutants in the active site of tyrosyl-t-RNA synthetase have alsobeen reported. Carter, P. J., et al. (1984) Cell 38, 835-840. In thisreport, the improved affinity of the previously described Thr51Promutant for ATP was probed by producing a second mutation in the activesite of the enzyme. One of the double mutants, Gly35/Pro51, reportedlydemonstrated an unexpected result in that it bound ATP in the transitionstate better than was expected from the two single mutants. Moreover,the author warns, at least for one double mutant, that it is not readilypredictable how one substitution alters the effect caused by the othersubstitution and that care must be taken in interpreting suchsubstitutions.

A mutant is disclosed in U.S. Pat. No. 4,532,207, wherein a polyargininetail was attached to the C-terminal residue of β-urogastrone bymodifying the DNA sequence encoding the polypeptide. As disclosed, thepolyarginine tail changed the electrophoretic mobility of theurogastrone-polyaginine hybrid permitting selective purification. Thepolyarginine was subsequently removed, according to the patentee, by apolyarginine specific exopeptidase to produce the purified urogastrone.Properly construed, this reference discloses hybrid polypeptides whichdo not constitute mutant polypeptides containing the substitution,insertion or deletion of one or more amino acids of a naturallyoccurring polypeptide.

Single and double mutants of rat pancreatic trypsin have also beenreported. Craik, C. S., et al. (1985) Science 228, 291-297. As reported,glycine residues at positions 216 and 226 were replaced with alanineresidues to produce three trypsin mutants (two single mutants and onedouble mutant). In the case of the single mutants, the authors statedexpectation was to observe a differential effect on Km. They insteadreported a change in specificity (kcat/Km) which was primarily theresult of a decrease in kcat. In contrast, the double mutant reportedlydemonstrated a differential increase in Km for lysyl and arginylsubstrates as compared to wild type trypsin but had virtually nocatalytic activity.

U.S. Pat. No. 4,760,025 discloses subtilisin mutants wherein a differentamino acid is substituted for the naturally-occurring amino acidresidues of Bacillus amyloliquifaciens subtilisin at positions +32,+155, +104, +222, +166, +64, +33, +169, +189, +217, or +156.

The references discussed above are provided solely for their disclosureprior to the filing date of the instant case, and nothing herein is tobe construed as an admission that the inventors are not entitled toantedate such disclosure by virtue of prior invention or priority basedon earlier filed applications.

Based on the above references, however, it is apparent that themodification of the amino acid sequence of wild type enzymes oftenresults in the decrease or destruction of biological activity. Moreover,these references do not address the mutation of the particular carbonylhydrolases disclose herein.

Accordingly, it is an object herein to provide carbonyl hydrolasemutants which have at least one property which is different from thesame property of the carbonyl hydrolase precursor from which the aminoacid of said mutant is derived.

It is a further object to provide mutant DNA sequences encoding suchcarbonyl hydrolase mutants as well as expression vectors containing suchmutant DNA sequences.

Still further, another object of the present invention is to providehost cells transformed with such vectors as well as host cells which arecapable of expressing such mutants either intracellularly orextracellularly.

SUMMARY OF THE INVENTION

The invention includes subtilisin-type carbonyl hydrolase mutants havinga different kcat, Km, and/or kcat/Km for various substrates as comparedto the precursor carbonyl hydrolase from which the mutant is derived.Such mutants consequently have a different reactivity with orspecificity for such substrates. The subtilisin-type carbonyl hydrolasemutants of the invention have an amino acid sequence not found in naturewhich is derived by the predetermined replacement of at least one aminoacid residue of a precursor carbonyl hydrolase with a different aminoacid. The mutant enzyme thus obtained is characterized by a shift incatalytic activity for at least one of two different substrates ascompared to the precursor enzyme.

The amino acid residue which is substituted in subtilisin comprisesproline 225 of the amino acid sequence of Bacillus amylolicuefacienssubtilisin and amino acid residues equivalent to said residue in otherprecursor subtilisin-type carbonyl hydrolases. Such othersubtilisin-type carbonyl hydrolases include subtilisin from sources suchas Bacillus subtilis and Bacillus licheniformis, each of which containproline at position 225. However, as will be described in more detailhereinafter, other subtilisin-type carbonyl hydrolases havingfunctionally or structurally equivalent residues to proline 225 insubtilisin are within the scope of the invention. Such substitutions maybe combined with the substitution, insertion or deletion of other aminoacid residues as described in U.S. Pat. No. 4,760,025 and the abovecross-referenced co-pending applications.

The invention also includes mutant DNA sequences encoding such carbonylhydrolase mutants. These mutant DNA sequences are derived from aprecursor DNA sequence which encodes a naturally occurring orrecombinant precursor carbonyl hydrolase. The mutant DNA sequence isderived by modifying the precursor DNA sequence to encode thesubstitution of one amino acid encoded by the precursor DNA sequence.These recombinant DNA sequences encode mutants having an amino acidsequence which does not exist in nature and at least one property whichis substantially different from the same property of the precursorcarbonyl hydrolase encoded by the precursor DNA sequence.

Further the invention includes expression vectors containing such mutantDNA sequences as well as host cells transformed with such vectors whichare capable of expressing said carbonyl hydrolase mutants.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the tertiary structure of subtilisin from Bacillusamyloliquefaciens subtilisin.

FIG. 2 is a stereo view of the α-helix associated with the catalyticSer22l in subtilisin.

FIG. 3A comprises the amino acid sequence for subtilisin form Bacillusamyloliquefaciens, Bacillus subtilis varI168 and Bacillus licheniformis.

FIG. 3B is a comparison of amino acid sequence of subtilisin fromB-amyloliquefaciens and thermitase.

FIG. 3C identifies the conserved residues in subtilisin.

FIG. 4 depicts the construction of mutants at position 225 insubtilisin.

FIG. 5A and 5B are a stereo view of residues 220 through 230 of wildtype and Pro 225A subtilisin, respectively.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have discovered that in vitro mutations at position 225 orequivalent amino acid residues of the non-human carbonyl hydrolasesubtilisin alter the catalytic activity, kcat, of the mutant enzyme fora target substrate as compared to the precursor enzyme from which it isderived, i.e. derived by the predetermined substitution of a differentamino acid for that in the precursor enzyme at position 225. The mutantcarbonyl hydrolases also may have a different Km and kcat/Km ratio andhence altered substrate specificity.

Non-human carbonyl hydrolases, recombinant carbonyl hydrolases,subtilisins, recombinant subtilisins, carbonyl hydrolase mutant,equivalent amino acid residues, prosequence, signal sequence, prepro,expression vector, host cells, operably linked, cassette mutagenesissubstrate specificity, multiple mutants and mutants at various otheramino acid residues are described in detail in application, Ser. No.035,652 filed Apr. 6, 1987, and such definitions are incorporated hereinby reference.

A change in substrate specificity is defined as a difference between thekcat/Km ratio for the precursor carbonyl hydrolase and that of thehydrolase mutant. The kcat/Km ratio is a measure of catalyticefficiency. Generally, the objective will be to secure a mutant having agreater (numerically large) kcat/Km ratio for a given substrate, therebyenabling the use of the enzyme to more efficiently act on a targetsubstrate. A substantial change in kcat/Km ratio is preferably at leasta 2-fold increase or decrease. However, smaller increases or decreasesin the ratio (e.g., at least 1.5-fold) are also considered substantial.An increase in kcat/Km ratio for one substrate may be accompanied by areduction in kcat/Km ratio for another substrate. This is a shift insubstrate specificity, and mutants exhibiting such shifts have utilitywhere the precursor hydrolase is undesirable, e.g. to prevent undesiredhydrolysis of a particular substrate in an admixture of substrates. Kmand kcat are measured in accord with known procedures, as described inEPO Publication No. 0130756 or as described herein.

A change in catalytic activity is defined as a difference between thekcat of the precursor carbonyl hydrolase for a particular targetsubstrate as compared to that of the mutant carbonyl hydrolase for thesame substrate. Generally, mutants having a greater (numerically larger)kcat for the target substrate are desired. Such mutants will have agreater catalytic activity with such substrates and consequently willpreferentially react with such substrates compared to the precursorenzyme A substantial change in kcat is preferably at least a two-foldincrease in kcat. However, smaller increases such as a 1.5-fold increasein kcat are significant provided the numerical value of kcat isrelatively large. Thus, a change in kcat from 500/sec to 750/sec is asubstantial change. A change from 5/sec to 7.5/sec, for example, is notnecessarily a substantial change in kcat.

A shift in catalytic activity is defined as the difference between thekcat ratio for two different substrates for the precursor carbonylhydrolase as compared to the mutant carbonyl hydrolase. Thus, forexample, the mutant subtilisin disclosed herein containing thesubstitution of alanine for proline at position 225 has a shift incatalytic activity, as compared to subtilisin not modified at position225, for ester and anilide substrates. A shift in catalytic activity isgenerally measured by determining the ratio of kcat for the precursorcarbonyl hydrolase for two different substrates. The kcat ratio is alsodetermined for the carbonyl hydrolase mutant. The kcat ratios for theprecursor and mutant enzymes are then compared. A substantial change inkcat substrate ratio is preferably at least a five-fold increase ordecrease in the kcat substrate ratio of the mutant enzymes as comparedto that of the precursor enzyme. However, smaller increases or decreasesin the kcat substrate ratio (e.g. at least about two-fold) are alsoconsidered substantial.

The three-dimensional structures of related subtilisins show a number ofhighly conserved structural features. The catalytic serine, Ser221, isfound near the beginning of a helix extending through the molecule, thesequence of which is conserved in evolutionarily related subtilisin-typeserine proteases. FIG. 1 shows the three dimensional x-ray structure ofsubtilisin from Bacillus amyloliquefaciens. The high-lighted segmentcomprises amino acid residues serine 221 through lysine 237. A stereoview of this segment isolated from the remainder of the molecule isshown in FIG. 2. There is a discontinuity at the junction of the 3₁₀helix 219-222 and α-helix 223-237 which is a direct consequence of thepresence of proline at position 225. The carbonyl oxygens of residues221 and 222 form hydrogen bonds with side-chain atoms rather than theamide nitrogens of residues 225 and 226 respectively. One of thesehydrogen bonds, between the carbonyl oxygen of Ser222 and the OG ofSer225, is in turn part of an extensive hydrogen bonding network. Bott(1988) supra. Proline is considered a strong helix breaker because itprevents the formation of a hydrogen bond between the carbonyl oxygen ofthe residue n-4 from the proline, in this case Ser221 and the nitrogenof proline 225. This results in the kink in the helix.

The conserved nature of this kink is further exemplified by the aminoacid sequence of various subtilisins. FIGS. 3A and 3B show the sequencehomology of subtilisins obtained from Bacillus amylolicuefaciensBacillus subtilis varI 168, Bacillus licheniformis and thermitase. FIG.3C identifies the various residues among the subtilisins which areconserved. As can be seen, the residues between gly219 and gly229 aretotally conserved except for the residues at position 224. Thus, themodifications described herein for subtilisin from Bacillusamyloliquefaciens are expected to produce similar results for othercarbonyl hydrolases of the subtilisin-type. Such subtilisin-likecarbonyl hydrolases accordingly are defined as any carbonyl hydrolasehaving a catalytic amino acid residue structurally or functionallyequivalent to the serine 221 in Bacillus amyloliquefaciens subtilisinwhich is located at the terminus of a α-helix. Examples of suchsubtilisin-type carbonyl hydrolases include subtilisin from Bacillussubtilis, Bacillus licheniformis, thermitase from Thermoretinomycesvalgaris and Proteinase K from fungi (Tritirachium album limber).

As disclosed herein, the substitution of proline 225 with alanineproduces a shift in catalytic activity of the mutant enzyme for estercontaining substrates as compared to anilide substrates which areclosely related to amide bonds. This residue is dispensable for the kinkpreceding the α-helix (residues 223-237). This single substitutioncauses a shift in catalytic activity toward ester substrates versesanilide substrates of almost 30-fold as compared to naturally occurringsubtilisin. It also results in a partial elimination of the kink so thatthe α-helix extends from residue 221-237. Modifications of this positionwith other amino acids are also expected to produce mutant enzymesdemonstrating a shift in catalytic activity for different substrates andsimilar structural consequences. Thus, other amino acids other thanalanine may be used to replace the proline at position 225. Such aminoacids preferably include leucine, methionine, glutamine, valine andserine, most preferably serine and leucine. These amino acids areexpected to extend the α-helix removing the discontinuity, or kink, in amanner analogous to the alanine substitution. Mutant enzymes containingsuch substituted amino acids at position 225 are also expected todemonstrate a shift in catalytic activity towards ester substrates ascompared to anilide or amide substrates.

Construction and Characterization of a Position 225 Mutant

The procedure used to substitute alanine for proline at position 225 inthe Bacillus amyloliquefaciens subtilisin gene is illustrated generallyin FIG. 4. Primer extension mutagenesis on a single stranded M13subclone using the mutagenic oligonucleotide in FIG. 4 was employed.Plasmid pPro225A was used to transform E-coli MM294.

Clones from this transformation were used to transform B. subtilis andtransformants were plated on Luria agar containing skim milk in order todetect protease secretion. One protease secreting transformant wasselected for enzyme purification and characterization and for sequenceanalysis to ensure that the coding sequences in and around the cassettewere correct.

Amino acid substitutions at position 225 alter the enzymesesterase/amidase activity. Table I compares the ratio of ester andanilide substrate kcat values for wild-type enzyme and subtilisin havingthe amino acid proline replaced with alanine at position 225.Measurements were performed using the substratessuccinyl-Ala-Ala-Pro-Phe-p-nitroanilade andsuccinyl-Ala-Ala-Pro-Phe-thiobenzyl ester at pH 8.6, 25 degrees Celsius.

                  TABLE I                                                         ______________________________________                                                     kcat ester substrate/                                            Enzyme       kcat anilide substrate                                           ______________________________________                                        Wild-type     33                                                              Ala-225      960                                                              ______________________________________                                    

In addition, Km was determined for these two substrates for subtilisinand the mutant containing the alanine substitution. The results of thatdetermination together with a calculation of kcat/Km is summarized inTable II.

                  TABLE II                                                        ______________________________________                                        Name   Substrate  pH      kcat   Km     kcat/Km                               ______________________________________                                        B.a.Subt                                                                             sAAPFpNA   8.60     50.   1.40 e-4                                                                             3.57 e5                               B.a.Subt                                                                             sAAPFsbz   8.60    1650.  6.80 e-5                                                                             2.43 e7                               A225   sAAPFpNA   8.60      3.6  6.20 e-4                                                                             5.75 e3                               A225   sAAPFsbz   8.60    3420.  3.99 e-4                                                                             8.57 e6                               ______________________________________                                    

One of the most striking structural features of subtilisins is a longhelix extending through the molecule comprising amino acids 221 through238. The first turn of this helix is interrupted by Pro 225 which causesa kink between the 3₁₀ helix 219-222 and the o-helix 223-237.

An increase in the effective dipole of the α-helix would increase theelectrophilicity of the oxyanion hole. The amide nitrogen of Ser221along with the n.sub.δ 2 of Asn155 form the oxyanion hole. An increaseof dipole strength would be expected by replacing Pro225 with alanine toallow hydrogen bond formation between Ser22l and the amide nitrogen ofalanine 225 thereby eliminating the kink in the α-helix. Increasing thedipole in the α-helix would increase the electrophilicity of the amidenitrogen of Ser221. Based on the expected increase in electrophilicity,the catalytic efficiency of the mutant enzyme for amide substratesshould be increased.

As can be seen, however, the substitution of proline for alanineresulted in just the opposite. The rate of peptide bond hydrolysisactually decreased (see kinetic data). The helix dipole therefore doesnot appear to be important for amidolysis. The rate of ester hydrolysis,however, increased for the Pro 225A mutant as compared to the wild-typesubtilisin.

FIG. 5 shows the difference in local structure between wild-type(Pro225) and mutant (Ala225) enzymes. As can be seen, the helix in theAla225 mutant extends from 221 rather than 223. The removal of the kinkessentially adds an additional turn of α-helix comprising residues 221through 224. Having described the preferred embodiments of the presentinvention, it will appear to those ordinarily skilled in the art thatvarious modifications may be made to the disclosed embodiments, and thatsuch modifications are intended to be within the scope of the presentinvention.

What is claimed is:
 1. An essentially pure subtilisin wherein the aminoacid residue equivalent to position +225 of Bacillus amyloliquifacienssubtilisin as shown in FIG. 3A, has been replaced with a differentnaturally occurring amino acid selected from the group consisting ofalanine, leucine, methionine, glutamine, valine, and serine, wherein theresulting mutated subtilisin is characterized by a shift in catalyticactivity of at least 1.5 fold, for at least one of two differentsubstrates as compared to a subtilisin wherein the position +225 residueis the same as that which is naturally occurring for said subtilisin. 2.A subtilisin of claim 1 wherein the resulting mutated subtilisin ischaracterized by a shift in catalytic activity for ester substrates ascompared to anilide substrates.
 3. The subtilisin of claim 1 whereinsaid replacement comprises Pro225Ala.